1. Field of the Invention
The present invention relates to a method of establishing an EBV-infected stomach cancer cell line from stomach cancer tissues infected with EBV, as well as EBV infecting cultured epithelial cells.
2. Description of the Related Art
EBV (Epstein-Barr Virus) is a DNA virus belonging to the family human herpes virus. When adult persons are first infected with it, infectious mononucleosis (IM) occur, but in Japan, the majority of persons are first infected latently with the virus at the infant stage, and through the life, latent infection continues. Accordingly, this virus has been suspected to be that causing many diseases including various cancers and autoimmune diseases, but there are many features unrevealed except that the connection with Barrkit [phonetic] lymphoma in Africa (Epstein M A, Barr Y M, Lancet Vol. 1:252-253, 1964) and upper pharyngeal cancer in the southern part of China (Pathnabathan R. et al., New Engl. J. Med. 333 (11):693-698, 1995) was proven.
In recent years, an EBV gene was detected in stomach cancer cells (Shousha S. et al., J. Clin. Pathol. 42:695-698, 1994) and further the EBV gene was found to be monoclonal (Imai S. et al., Proc. Natl. Acad. Sci. USA, 91:1931-1935, 1994), and since it was suggested that the EBV gene may be involved in the canceration process, it came to be thought that EBV is involved in at least a part of stomach cancers.
At present, no diagnostic method has been established for specifying whether EBV is involved in canceration in each patient with stomach cancer, so it is not revealed whether the stomach cancer cancerated by EBV, as compared with other stomach cancers, has distinct characters such as easiness of metastasis, rapidness of the progress, drug sensitivity etc. While establishment of a diagnostic method therefor is desired, establishment of a therapeutic method and particularly development of chemotherapy for the stomach cancer in which EBV has been involved are an important task. For establishment of the diagnostic method and development of the chemotherapy, establishment of a stomach cancer cell line cancerated by EBV is essential.
Recently, the present inventors reported establishment of epithelial cells GT38 & GT39 derived from stomach normal tissues in patients with stomach cancer (Tajima M. et al., Jpn. J. Cancer Res. 89:262-268, 1998). It was surprising that EBV whose natural host is lymphoid B cells infects epithelial cells to produce EBV-related antigens though in a small amount, but for establishment of the diagnostic method and development of the chemotherapy, it was desired to establish a stomach cancer cell line which was established not from normal tissues but from cancer tissues, that is, a stomach cancer cell line which certainly underwent in vivo canceration.
Further, if canceration occurs due to a product of the EBV gene, a new type of anticancer drug inhibiting the mechanism of canceration can be developed. To analyze the mechanism of canceration of stomach cancer by EBV, it would be necessary to infect epithelial cells with EBV in order to analyze which gene in EBV is involved in the canceration.
However, it was not possible to efficiently infect cultured epithelial cells with EBV in vitro, although the relationship between EBV and the stomach cancer was estimated since the EBV gene was also found in vivo in epithelial cells of stomach cancer cells etc. (Shousha S. et al., J. Clin. Pathol. 47:695-698, 1994). Up to now, it is has been reported that genetic recombinant EBV was prepared for infecting cultured epithelial cells with EBV (Borza C M et al., J. Virol. 72 (9):7577-82, 1998; Imai S. et al., J. Virol. 72 (5):4381-8, 1998). In these reports, a method of amplifying and capturing a very rare phenomenon, that is, a method of infecting the cells with EBV having a drug resistance gene (Neo.sup.r) inserted into it followed by selection with the drug (neomycin), is used. For example, in the report of Imai in 1998, it is reported that upon infection of a culture supernatant (400,000 cells), 32 cells at the maximum were successfully infected, and there is no guarantee that such a rare phenomenon reflects naturally occurring infection with EBV. Up to now, there is no report on efficient infection of epithelial cells with EBV whose gene is not subjected to recombination, but a system for infection of epithelial cells with EBV whose gene is not modified is necessary to analyze the mechanism of actually occurring in vivo canceration by EBV.
In epidemiological study, it was found that the antibody titer toward EBV-related antigens i.e. viral capsid antigen (VCA) and latent membrane protein (LMP) in patients with stomach cancer is higher than in healthy persons (Masako Tajima, "Rinsho To Virus" (Clinics and Virus), Vol. 25, No. 3:169-176, 1997), and the relationship between the stomach cancer and EBV was thus supported, and simultaneously the worth of diagnosis of the antibody titer toward EBV-related antigens came to attract attention.
To utilize the EBV-related antigens in diagnosis of stomach cancer, stable supply of EBV-related antigens is essential. In consideration of post translational modification of proteins, cells producing EBV-related antigens are desirably epithelial cells, particularly stomach cancer cells. However, generally known EBV-infected cells such as B95-8 and Daudi are lymphoid cells and have the problem of low production of VCA and LMP. Further, GT38 & GT39 established from normal tissues in patients with stomach cancer, reported recently by the present inventors (Tajima M. et al., Jpn. J. Cancer Res. 89:262-268, 1998), had the problem of low production of VCA and LMP, as well. Further, depending on culture conditions, differentiation and induction occurred in these cells, so there was the problem of unstable production of EBV-related antigens. It was desired that a stomach cancer cell line stably producing EBV-related antigens is established from stomach cancer tissues considered to have stable traits at the final stage of canceration.